The present invention relates generally to characterizing responses of ligand-functionalized surfaces and, more particularly, to microfluidic probes and the dispensation of segmented liquid flows by such microfluidic probes to characterize such responses.
A current standard technique used in measurements of biomolecular interactions and binding kinetics is the surface plasmon resonance (SPR) based analysis. This technique enables measurements of reaction kinetics of label-free reactants. It has, however, several disadvantages, among which include the following:
The distance between the bound receptor and the surface is limited to distances that are on the order of 200 nm. This limits the types of analyzed reactants to small molecules and does not allow accurate measurements using larger surface-bound ligands, such as cells (cell interactions can only be measured by using cells as the bulk analyte);
The SPR technique lacks sensitivity when measuring low molecular weight adsorbates;
SPR further requires a complex substrate, e.g., comprising a glass-metal film (typically the sensor chip is a gold-coated glass slide);
Due to its detection mechanism, the SPR method is limited to measurements of unique interactions and does not allow simultaneous kinetic measurements of multivalent interactions and competitive reactions;
The SPR technique requires specialized and expensive equipment, which may not always be affordable; and
SPR is highly susceptible to temperature. Variations in temperature of less than 1 Kelvin can significantly impact the sensitivity of SPR.
An advance on the traditional SPR systems is the XPR system, or multiplexed SPR, which involves an array system of orthogonal and parallel channels. Different concentrations or analytes can thus be injected into each channel to create multiple pairings of reagents and conditions. While this improvement enables multiplexing in SPR, each measurement in the array corresponds to a unique interaction, i.e., measurements of competitive or multivalent interactions are not possible.
Labeled biomolecules (e.g., fluorescent, radioactive, chemiluminescent, etc.) are widely used in various biochemistry applications. Direct signal measurements can be performed using standard equipment, such as fluorescence microscopes and detectors, and therefore are commonly available in biology and research laboratories. However, real-time signal measurement of interactions between such molecules is limited by the background signal of the labeled analyte in the bulk, which is significantly higher than the signal of the bound analyte molecules.
Aside from SPR and XPR system, one knows microfluidic systems and related techniques, which deal with the behavior, precise control and manipulation of small volumes of fluids that are typically constrained to micrometer-length scale channels and to volumes typically in the sub-milliliter range. Prominent features of microfluidics originate from the peculiar behavior that liquids exhibit at the micrometer length scale. Volumes well below one nanoliter can be reached by fabricating structures with lateral dimensions in the micrometer range. Reactions that are limited at large scales (by diffusion of reactants) can be accelerated. Finally, parallel streams of liquids can possibly be accurately and reproducibility controlled, allowing for chemical reactions and gradients to be made at liquid/liquid and liquid/solid interfaces
Microfluidic devices generally refer to microfabricated devices, which are used for pumping, sampling, mixing, analyzing and dosing liquids. A microfluidic probe is a device for depositing, retrieving, transporting, delivering, and/or removing liquids, in particular liquids containing chemical and/or biochemical substances. For example, microfluidic probes can be used in the fields of diagnostic medicine, pathology, pharmacology and various branches of analytical chemistry. Microfluidic probes can also be used for performing molecular biology procedures for enzymatic analysis, deoxyribonucleic acid (DNA) analysis and proteomics.